A HER-2 binding antagonist is described and provided. Specifically, intron retention has generated a novel HER-2 antagonist polypeptide that binds to the HER-2 receptor.
The HER-2/neu (erbB-2) oncogene encodes a receptor-like tyrosine kinase (RTK) that has been extensively investigated because of its role in several human carcinomas (Hynes and Stem, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994) and in mammalian development (Lee et al., Nature 378:394-398, 1995). The sequence of the HER-2 protein was determined from a cDNA that was cloned by homology to the epidermal growth factor receptor (EGFR) mRNA from placenta (Coussens et al., Science 230:1132-1139, 1985) and from a gastric carcinoma cell line (Yamamoto et al., Nature 319:230-234, 1986). The HER-2 mRNA was shown to be about 4.5 kb (Coussens et al., Science 230:1132-1139, 1985; and Yamamoto et al., Nature 319:230-234, 1986) and encodes a transmembrane glycoprotein of 185 kDa in normal and malignant human tissues (p185HER-2) (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994). The function of the HER-2 gene has been examined mainly by expressing the cDNA corresponding to the 4.5 kb transcript in transfected cells and from the structure and biochemical properties of the 185 kDa protein product. P185HER-2 consists of a large extracellular domain, a transmembrane segment, and an intracellular domain with tyrosine kinase activity (Hynes and Stern, Biochim. et Biophys. Acta 1198:165-184, 1994; and Dougall et al., Oncogene 9:2109-2123, 1994). Overexpression of p185HER-2 causes phenotypic transformation of cultured cells (DiFiore et al., Science 237:178-182, 1987; and Hudziak et al., Proc. Natl. Acad. Sci. USA 84:7159-7163, 1987) and has been associated with aggressive clinical progression of breast and ovarian cancer (Slamon et al., Science 235:177-182, 1987; and Slamon et al., Science 244:707-712, 1989). p185HER-2 is highly homologous to the EGFR. However, a ligand that directly binds with high affinity to p185HER-2 has not yet been identified. Moreover, the signaling activity of HER-2 may be mediated through heterodimerization with other ligand-binding members of the EGFR family (Carraway and Cantley, Cell 78:5-8, 1994; Earp et al., Breast Cancer Res. Treat. 35:115-132, 1995; and Qian et al., Oncogene 10:211-219, 1995).
Divergent proteins, containing regions of the extracellular domains of HER family RTKs, are generated through proteolytic processing of full length receptors (Lin and Clinton, Oncogene 6:639-643, 1991; Zabrecky et al., J. Biol. Chem. 266:1716-1720, 1991; Pupa et al., Oncogene 8:2917-2923, 1993; Vecchi et al., J. Biol. Chem. 271:18989-18995, 1996; and Vecchi and Carpenter, J. Cell Biol. 139:995-1003, 1997) and through alternative RNA processing (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990; Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993; and Lee and Maihle, Oncogene 16:3243-3252, 1998). The extracellular domain of p185HER-2 is proteolytically shed from breast carcinoma cells in culture (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990; Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993; and Lee and Maihle, Oncogene 16:3243-3252, 1998), and is found in the serum of some cancer patients (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992) where it is may be a serum marker of metastatic breast cancer (Leitzel et al., J. Clin. Oncol. 10:1436-1443, 1992) and may allow escape of HER-2-rich tumors from immunological control (Baselga et al., J. Clin. Oncol. 14:737-744, 1966; and Brodowicz et al., Int. J. Cancer 73:875-879, 1997).
A truncated extracellular domain of HER-2 is also the product of a 2.3 kb alternative transcript generated by use of a polyadenylation signal within an intron (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993). The alternative transcript was first identified in the gastric carcinoma cell line, MKN7 (Yamamoto et al., Nature 319:230-234, 1986; and Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993) and the truncated receptor was located within the perinuclear cytoplasm rather than secreted from these tumor cells (Scott et al., Mol. Cell. Biol. 13:2247-2257, 1993). However, no particular therapeutic, diagnostic or research utility has been ascribed to this truncated extracellular domain polypeptide. A truncated extracellular domain of the EGFR, generated by alternative splicing (Petch et al., Mol. Cell. Biol. 10:2973-2982, 1990) is secreted, exhibits ligand-binding, and dimerization properties (Basu et al., Mol. Cell. Biol. 9:671-677, 1989), and may have a dominant negative effect on receptor function (Basu et al., Mol. Cell. Biol. 9:671-677, 1989; and Flickinger et al., Mol. Cell. Biol. 12:883-893, 1992).
Group I receptor tyrosine kinases including the EGF-receptor (HER-1, erbB-1), HER-2 (erbB-2), HER-3 (erbB-3), and HER-4 (erbB-4) are widely expressed in epithelial, mesenchymal, and neuronal tissues and play fundamental roles in proliferation and differentiation. With the exception of p185HER-2, receptor tyrosine kinases are activated by binding to a variety of EGF-related growth factors. Ligand binding is coupled to receptor dimerization, tyrosine autophosphorylation, and signal activation. Independently of a specifically binding growth factor, p185HER-2 dimerizes with itself or is recruited as the preferred heterodimer partner where it transactivates receptor family members.
Enhanced amounts of group receptors at the cell membrane occurs frequently in human carcinomas. This elevation in number of receptors is likely to favor the formation of receptor oligomers resulting in amplified signaling. The EGF-receptor and p185 HER-2 have been most frequently and clearly associated with human malignancies. HER-2 is overexpressed in breast, ovarian, gastric, and endometrial carcinomas Elevated levels of p185HER-2 in 25-30% of breast and ovarian cancers predicts significantly lower survival rates and shorter time to relapse. Amplification and alteration of the EGF-receptor gene is often observed in squamous cell carcinoma of the lung (Pavelic et al., 1993) and in glial tumors (Libermann et al., 1985), particularly in glioblastoma, the most malignant glial tumor.
There have been extensive efforts directed toward defining the structure and function of the group I receptor extracellular domains in the interests of understanding the mechanism of receptor activation and in blocking receptor action at the cell surface. Receptor mutants consisting of the extracellular domain and a membrane anchor, in the absence of the cytoplasmic domain, are capable of dimerizing (Lemmon et al., 1997; Tzahar et al., 1997;Tanner and Kyte 1999) and forming kinase inactive complexes with cell surface receptors (Greene). The ectodomains of group I receptors have been divided into subdomains I, beginning at the N-terminus, through IV ending at the juxtamembrane position. Domains II and IV contain multiple cysteine residues that are conserved amongst the four group I receptors. Subdomains I and II appear to be a repeating unit of III and IV that may have arose by a gene duplication event (Ullrich et al., 1984). Deletion of subdomains I and II from the EGF receptor results in constitutive dimerization and oncogenic transformation in a ligand-independent fashion (Hayely et al., 1989; Carter and Kung 1994; Qian et al., 1994; Moscatello et al., 1996), and allows ligand independent heterodimer formation with the membrane anchored p185neu ectodomain mutant (Greene). While subdomain III contains the high affinity ligand binding site as shown for EGF binding to the EGF receptor (Wu et al., 1990 Woltjer et al., 1992 Lax et al., 1989; 1991), subdomain I has been suggested to serve as a low affinity site that is promiscuous in its ligand recognition (Lax et al., 1989; 1991 Tzahar et al., 1997). According to this model EGF-like ligands are bivalent with a high affinity site that binds to the direct receptor in subdomain III and a second, low affinity site with broad specificity for subdomain I that prefers interaction with p185HER-2, thereby explaining the status of p185HER-2 as the preferred dimer partner. Taken together these results suggest that subdomains I and II may exert a negative constraint on dimerization in the absence of ligand and could be important for recruitment of receptors into heterodimers.
Monoclonal antibodies against the ectodomains of p185HER-2 and the EGF-receptor have been shown to be effective in limiting growth of tumors. These antibodies bind to their receptor targets with high affinity and specificity and their toxicity is low. The mechanisms underlying the antitumorigenic effects of antibodies are unclear. The rhuMAb4D5 (HERCEPTIN®) antibody may act by downregulation of p185HER-2 at the cell surface (Hurwitz et al., 1995), which causes a reversible cytostatic effect on HER-2 mediated cell growth. Systemic administration of the monoclonal antibody rhuMAb4D5 (HERCEPTIN®) has been shown to have therapeutic efficacy, since it increases the time to recurrence in a subset of patients with metastatic breast cancer. High affinity humanized, monoclonal antibodies against the EGF receptor have also been used as antitumor agents. While the molecular mechanisms underlying the activity of EGF receptor antibodies remain elusive, those tested compete with growth factor binding. Antibody strategies that target p185HER-2 and the EGF-receptor, as well as heterodimers between these two receptors, have also been attempted. Preliminary evidence suggests that targeting both receptors may significantly augment antiproliferative effects.
Mutant receptors consisting of ectodomains have proved to be effective in inhibition of tumorigenesis. The membrane-anchored ectodomain of p185 neu, ectopically expressed in cells, functions as a dominant negative inhibitor based on its ability to dimerize with the ectodomains of group I receptors forming a kinase-inactive complex. P185neu ectodomain mutants are capable of specific inhibition of p185HER-2 homodimer signaling as well as trans-inhibition of EGF receptor signaling. Since p185HER-2 is the preferred heterodimer partner of group I RTKs, then the p185-ectodomain is capable of suppressing the activation of all group I receptors. However, membrane anchoring of ectodomain mutants is required to exert a dominant negative effect since interactions between soluble ectodomains and cell surface receptors are too weak to achieve complex formation.